Process and compositions for determination of uric acid in blood serum



Sept. 15, 1970 ABJOFBA/Vff J. J. MORAN PROCESS AND COMPOSIIIONS FOR DETERMINATION OF 0032 Z.50 19'Z 61 0/2 2 X51 97 /0 0197;

(/R/C A017 f0fir7 J Mara/7 INVENTOR. 16: F M BY /fi 61/4/- United States Patent Office 3,528,777 Patented Sept. 15, 1970 U.S. Cl. 23230 14 Claims ABSTRACT OF THE DISCLOSURE A process of detecting uric acid in blood serum containing protein by mixing a sodium carbonate solution and an alkyl sulfate surfactant with a dilute solution of the blood serum, adding thereto a phosphotungstic acid solution and a dilute solution of hydrazine dihydrochloride, determining the optical absorbance of the resulting products, and then comparing that optical absorbance with the optical absorbance of samples of uric acid standard containing known quantities of uric acid which have been treated in the same manner; and the compositions used in such process.

BACKGROUND OF THE INVENTION The field of art to which the invention pertains is the process and compositions for the determination of uric acid in blood. Human blood normally contains small quantities of uric acid and medical science has long recognized the usefulness of tests for measuring the amounts or change of uric acid in blood as an aid in diagnosing certain medical conditions such as gout.

One of the most Widely used methods of determining uric acid in blood is the test performed on blood serum described by Caraway at pages 239241 of Standard Methods of Clinical Chemistry, volume IV by the American Association of Clinical Chemists published 1963 by Academic Press, Inc., 111 Fifth Ave., New York 10003. That test is a colorimetric determination of uric acid in blood based on the reduction of phosphotungstate by uric acid in alkaline solution. In that method, which is believed to have been originally developed by Folin and Denis, a tungstic acid solution is prepared by mixing distilled water, sodium tungstate, 85% o-phosphoric acid and sulfuric acid. 9.0 ml. of tungstic acid solution is added to 1.0 ml. of blood serum which results in a precipitation of the proteins in the blood serum. The precipitate is removed by filtration or centrifuging. ml. of the clear supernatant liquid or filtrate is placed in a test tube or cuvette and 5 ml. of distilled water is placed in a second tube for a blank. To each tube is added 1.0 ml. of a dilute sodium carbonate solution and the material is mixed. The tubes are allowed to stand for ten minutes and then to each tube is added 1.0 ml. of dilute phosphotungstic acid, the material is mixed immediately, and the tubes are then allowed to stand for approximately thirty minutes. During this thirty minute period, the clear liquid in the tube containing the treated serum turns light blue in color. The liquid in the other tube remains clear. The optical absorbance or optical density of the unknown is then measured against the blank in a spectrophotometer usually set at a wave length of about 700 m The optical absorbance of the unknown is then compared with a previously prepared curve of concentration of uric acid versus optical absorbance which curve has been prepared from varying concentrations of uric acid standard containing known amounts of uric acid that has been treated identically with the unknown sample. By comparing the optical absorbance of the unknown sample with this curve, the amount of uric acid in the unknown sample can be read directly.

Simoes in Archives of Portuguese Biochemistry, volume 6, pages 192-197 (l96263) suggests the use of hydrazine or hydrazine hydrate with the phosphotungstic acid solution to intensify the blue color formed by the addition of the phosphotungstic acid solution.

The phosphotungstic acid solution used in the conventional process described above and in the present invention is the same and preferably is prepared by dissolving 50 grams of molybdate-free sodium tungstate in 400 ml. of distilled water in a 1,000 ml. flask fitted with a reflux condenser. 40 ml. of o-phosphoric acid is added, the condenser is attached, and the material is boiled gently for two hours. The contents are then cooled to room temperature and transferred with rinsing to a 500 ml. volumetric flask and diluted to the 500 ml. volumetric mark with water. This material is then diluted with nine parts of water to make the phosphotungstic acid solution used.

The uric acid standard used in the conventional process described above is prepared as follows: 1.0 gram of uric acid is transferred to a 1,000 ml. volumetric flask. 0.60 gram of lithium carbonate is dissolved separately in 150 ml. of warm Water, filtered, and the filtrate heated to approximately 60 C. The slight turbidity that develops at this point is ignored. The Warm lithium carbonate solution is added to the flask and mixed until the uric acid is completely dissolved. Solution should be complete in five minutes; the flask may be warmed under running hot water if necessary, then held under running cold water to cool. The solution usually remains slightly turbid. 20 ml. of 40% formaldehyde is added and the contents are diluted to about 500 ml. with water. Next there is added slowly and with mixing 25 ml. of 1 N sulfuric acid (prepared by diluting concentrated acid 1:36), the contents are then diluted to the 1,000 ml. mark with water and mixed well. This gives a uric acid standard of mg. per 100 ml. This may be diluted with distilled water to give the desired concentration of uric acid standard which in the conventional test is 0.5 mg. per 100 ml.

The present invention eliminates the removal of protein from the blood serum and permits the use of much smaller samples of blood serum in making the tests.

SUMMARY OF THE INVENTION It is a general object of the present invention to provide a process for the detection of uric acid in blood and a test system or compositions useful in that process.

A more particular object of the present invention is to provide such a process in which the test is used on a relatively small sample of blood serum from which the protein has not been removed.

A still further object of the present invention is to provide such a process utilizing sodium carbonate solution and dilute phosphotungstic acid solution of the type previously described herein.

Other and further objects, features and advantages will be apparent from the following description of the presently preferred examples of the present invention given for the purpose of disclosure and taken in conjunction with the accompanying drawing.

The present invention is based upon a discovery that the amount of uric acid in blood may be determined by testing blood serum containing protein and that only a relatively small sample of such serum need be used if a small amount of an alkyl sulfate surfactant is added to the serum prior to or at approximately the same time the sodium carbonate solution is added and if a dilute solution of hydrazine dihydrochloride (N H -2HCl) is added at approximately the same time the phosphotungstic acid solution is added.

The conventional method using the carbonate and phosphotungstic acid requires, as set forth above, the removal of the protein from the serum asthe presence of protein in the conventional method causes a cloudiness or turbidity which makes the test difficult and inaccurate. Not only is the removal of the protein a timeconsuming, hence expensive, step when many tests are run but it introduces the possibility of additional human error. When the alkyl sulfate surfactant of the present invention is added to the protein-containing serum, the later or simultaneous addition of the sodium carbonate prevents cloudiness and the serum remains clear.

With the present invention, a 0.1 ml. sample of serum is normally used as compared with the 1.0 ml. sample used in the conventional carbonate-phosphotungstic process. It requires about 3.5 ml. of whole blood to make 1.0 ml. of serum and since clinical tests are often run in duplicate for accuracy and when blood is removed from a patient for testing, it is usually removed for testing for more than one condition, the amount of serum needed for any test becomes important. In the conventional carbonate-phosphotungstic acid test, the com-paratively large amount of serum is necessary because the blue color that results after the addition of the phospho tungstic acid solution is so light in color that the larger volume of liquid is necessary in order to have an absorbance that can be read accurately on a conventional spectrophotometer.

The addition of the hydrazine dihydrochloride in some way intensifies the color that results when the phosphotungstic acid solution is added. The use of hydrazine and hydrazine hydrate will also intensify the color but not nearly as much as hydrazine dihydrochloride, and with the small serum samples used in the present invention, hydrazine and hydrazine hydrate will not intensify the color sufliciently to give an accurate reading on a conventional spectrophotometer.

BRIEF DESCRIPTION OF THE DRAWING The drawing is a curve of absorbance versus concentration in milligram percent of uric acid determined from uric acid standards of known concentration of uric acid.

DESCRIPTION OF THE PREFERRED EMBODIMENT In the preferred embodiment of the present invention, a standard curve such as that shown in the drawing is determined in the following manner. 1.0 ml. of 100 mg. percent uric acid standard (prepared as previously described) is mixed with 9.0 ml. of distilled water. Five test tubes are marked blank, 2.5, 5, 7.5 and 10. Into the test tube marked blank is placed 4.0 ml. of distilled water, into the test tube marked 2.5 is placed 1.0 ml. of the diluted standard and 3 ml. of water, into the test tube marked 5 is placed 2.0 ml. of diluted standard and 2.0 ml. of distilled water, into the test tube marked 7.5 is placed 3.0 ml. of diluted standard and 1.0 ml. of distilled water, and into the test tube marked is placed 4.0 ml. of diluted standard and no distilled water. Each tube now contains 4.0 ml. of working standard equivalent to 0, 2.5, 5, 7.5 and 10 mg. percent uric acid.

Into five test tubes marked 0, 2.5, 5, 7.5 and 10, place 0.1 ml. sample corresponding to the five Working standards. In timed thirty second sequence, into each of the five last-mentioned test tubes is placed 3.0 ml. of a carbonate reagent which is a mixture of 5% sodium carbonate and 0.2% alkyl sulfate surfactant in distilled water. Immediately after each addition, the contents are mixed by gentle swirling. The contents of each of the test tubes at this time is a clear liquid.

Exactly twenty minutes after the addition of the car bonate reagent to the first test tube and in the same timed thirty second sequence, exactly 3.0 ml. of phosphotungstic reagent is added to each test tube, the contents are mixed by shaking, and then the tubes are placed in a rack and allowed to stand at room temperature. The phosphotungstic reagent that is added is a mixture of 0.1% bydrazine dihydrochloride with the balance being the phosphotungstic acid solution previously described herein.

Exactly twenty minutes after the addition of the phosphotungstic reagent to the first test tube, and in the same timed thirty second sequence, the optical absorbance or density of the standards is read against the blank at 580 mu. in a spectrophotometer. Thereafter a standard curve is prepared of the optical absorbance versus con centration of the standards by plotting concentrations as the abscissa and optical absorbance as the ordinate. Such a standard curve is shown in the drawing.

Either simultaneously with the steps recited above for preparing the standard curve or at some other time, the protein-containing blood serum sample to be tested for uric acid content is identically treated, that is 0.1 ml. of it is placed in a test tube, 3.0 ml. of sodium carbonate reagent is added and mixed, twenty minutes later the phosphotungstic reagent is added and mixed, and twenty minutes after the addition of the phosphotungstic reagent, the optical absorbance is read. When this optical absorb ance is determined, the uric acid content in milligram percent is then read directly from the standard curve.

The uric acid content of other samples of proteincontaining blood serum may be similarly determined using the same standard curve.

For convenience in use, the alkyl sulfate surfactant is mixed with the sodium carbonate prior to carrying out the process, but this alkyl sulfate surfactant may be added to the test tubes prior to the addition of the sodium carbonate if desired. The preferred alkyl sulfate surfactant is sold under the trade name Teepol by Shell Chemical Co., P.O. Box 2392, Church Street Station, New York, N.Y. 10008 and is a sodium secondary alkyl sulfate having the general formula:

wherein R and R are alkyl groups of different size. This surfactant has a pH between 8.5 and 9, is a clear amber liquid, and has a specific gravity of 1.08.

The time between the addition of the carbonate reagent and the addition of the phosphotungstic reagent will vary if other concentrations of sodium carbonate are used. Apparently the sodium carbonate causes an oxidation of some of the interferring substances such as purine rings related to uric acid and other substances. If there is an insufiicient amount of time between the addition of the carbonate reagent and the addition of the phosphotungstic reagent for the reaction to take place with the sodium carbonate, elevated values of uric acid will be obtained. The amount of time necessary between the addition of a carbonate reagent of some other concentration and the addition of the phosphotungstic reagent can be determined by running tests of time against known concentrations of uric acid to determine the minimum amount of time necessary. The twenty minutes set forth in the preferred example above for Waiting between the adding of the carbonate reagent and the addition of the phosphotungstic reagent is a minimum and if desired the time may be extended.

With the particular phosphotungstic reagent set forth above in the preferred example, if thereading of the absorbance is delayed an appreciable time after the addition of the phosphotungstic reagent, a precipitation will appear in the test tube which will cause inaccuracies in the test. If the absorbance is determined in an appreciable time earlier than twenty minutes, the color intensity will be changing while the absorbance is being determined and this introduces inaccuracies in the test.

If other concentrations of phosphotungstic acid solution in the phosphotungstic reagent set forth above are used, it will affect the time at which readings of absorbance should be taken. If other concentrations of the phosphotungstic acid solution are used, then the time between the addition of the phosphotungstic reagent and the reading of the color in the spectrophotometer can be determined by plotting color development versus time on a spectrophotometer equipped with a recorder. The plot of that curve will show an increase in color as time increases until a plateau is reached at which the color intensity does not change appreciably with change in time and it is at a time within that plateau at which the absorbance should be read but before the precipitation occurs. The twenty minutes set forth above between the addition of the phosphotungstic reagent and the reading of the absorbance is in that plateau for the preferred concentration of phosphotungstic acid.

Both the alkyl sulfate surfactant and the hydrazine dihydrochloride are necessary for the process of the present invention. If the alkyl sulfate surfactant is not used, a turbidity will often result when the sodium carbonate is added which will make the absorbance reading too inaccurate to be of use with or without the later addition of the hydrazine dihydrochloride. If the alkyl sulfate surfactant is used but the hydrazine dihydrochloride is not used, the blue color that results after the addition of the phosphotungstic acid will be too pale to give an accurate reading.

Of course uric acid standards other than the one previously disclosed herein may be used such as the Monitrol I, Normal Clinical Chemistry Highland, and Versatol natural and synthetic blood serums described in the following example.

Set forth below is a specific example of the use of the process and test system or compositions of the present invention.

EXAMPLE In this example, duplicate tests were run on the standards to prepare the standard curve and on three test samples simultaneously, otherwise the procedure followed was exactly that set forth in the preferred embodiment. In preparing the data for the standard curve, in addition to the five test tubes being marked 0, 2.5, 5, 7.5 and and having placed therein 0.1 ml. of working standard equivalent to 0, 2.5, 5, 7.5 and 10 mg. percent uric acid respectively, another set of five test tubes was identically marked and prepared. The three samples to be tested were 1) a blood serum without the protein removed sold under the trade name Monitrol I by Dade Reagents Incorporated, Miami, Fla., (2) a blood serum without the protein removed sold under the trade name Normal Clinical Chemistry Highland by Highland, Incorporated, Los Angeles, Calif. and (3) a synthetic blood serum containing protein sold under the trade name Versatol by Warner-Shilcott, Morris Plains, N.J..

The absorbance was read in a Coleman spectrophotometer and the standards gave at 2.5 mg. percent readings of .239 and .238 absorbance units, at 5.0 mg. percent gave .460 and .455 absorbance units, at 7.5 mg. percent gave .600 and .610 absorbance units, and at 10.0 percent gave .678 and .680 absorbance units. The average of each of these two absorbance units readings for each of these milligram percents was used to plot the standard curve.

The two tests run on the Monitrol I gave absorbance units of .452 each and reading the uric acid concentration from the curve at .452 absorbance units give a concentration of 5.0 mg. percent. The assay value given by the manufacturer for uric acid content of that sample of Monitrol I was 5.1 mg. percent. The Normal Clinical Chemistry Highland sample had absorbance readings of .418 and .422 which values read from the curve give respectively 4.6 mg. percent and 4.7 mg. percent. The

manufacturers assay value of uric acid for this sample was 4.7 mg. percent. The Versat-ol sample absorbance units readings were .308 and .310 which from the curve both give 3.2 mg. percent as compared to the manufacturers assay value of 3.0 mg. percent. All of these percentages are well within the expected tolerance of such tests.

From the foregoing discussions, examples and description of the invention, it is apparent that the objects set forth herein as well as others have been achieved. Those skilled in the art will recognize that the principle of this invention may be applied in several ways, only a few of which have been exemplified here specifically. Accordingly, the invention is to be limited only by the spirit thereof and the scope of the appended claims.

What is claimed is:

1. In the process for detecting uric acid in blood serum by mixing a sodium carbonate solution with a dilute sample of the blood serum, adding thereto a phosphotungstic acid solution, determining the optical absorbance of the resulting products, and then comparing that optical absorbance with the optical absorbance of samples of uric acid standard containing known quantities of uric acid which samples of uric acid standard have been treated in the same manner, the improvement comprising (a) performing the process on blood serum containing protein,

(b) mixing an alkyl sulfate surfactant with the sample at least as early in time as the addition of the sodium carbonate solution, and

(c) adding and mixing a dilute solution of hydrazine dihydrochloride with the phosphotungstic acid solution.

2. The process of claim 1 in which the alkyl sulfate surfactant is a sodium secondary alkyl sulfate.

3. The process of claim 1 in which the alkyl sulfate surfactant is a sodium secondary alkyl sulfate having the general formula:

C JOQONa Rf wherein R and R are different size alkyl groups.

4. The process of claim 2 in which the alkyl surfactant is added in the strength of an approximately 0.2% solution.

5. The process of claim 1 in which the hydrazine dihydrochloride is added in the strength of an approximately 0.1% solution.

6. The process of claim 2 in which the hydrazine dihydrochloride is added in the strength of an approximately 0.1% solution.

7. In a process for detecting uric acid in blood serum by comparing the optical absorbance of treated samples of uric acid standard containing known quantities of uric acid with the optical absorbance of a treated sample of such serum, the improvement comprising the steps of (a) placing in a transparent container a measured amount of blood serum containing protein,

-(b) placing and mixing in such container a measured amount of an alkyl sulfate surfactant,

(c) placing and mixing in the container a measured amount of a dilute solution of sodium carbonate, step (b) being performed at least as early in time as step (c),

(d) after the completion of the reaction caused by step (c), placing and mixing a measured amount of phosphoric acid solution and of a dilute solution of hydrazine dihydrochloride in the container,

*(e) measuring the optical absorbance of the liquid in the container after the completion of the reaction caused by step (d) and prior to the formation of a precipitate,

(f) placing in each'of several transparent containers different concentrations of uric acid standard containing a known concentration of uric acid, the volume of material in each container being the same as the volume of material in the container of p (g) performing steps (b) through (e) with respect to each of the transparent containers of step (f),

(h) plotting a curve of optical absorbance versus concentration of uric acid in each of the containers of step, (f), and

(i) comparing the optical absorbance of step (e) with the curve of step (h) to determine the concentration of uric acid in the blood serum, steps (a) through (c) being performed in any order with respect to steps (f) through (h).

8. The process of claim 7 in which the amount of blood serum used in step (a) is approximately 0.1 ml.

9. The process of claim 7 in which the alkyl sulfate surfactant is a sodium secondary alkyl sulfate.

10. In a process for detecting uric acid in blood serum by comparing the optical absorbance of treated samples of uric acid standard containing known quantities of uric acid with the optical absorbance of a treated sample of such serum, the improvement comprising the steps of (a) placing in a transparent container 0.1 ml. of

blood serum containing protein,

(b) placing and mixing in the container 3.0 ml. of a mixture of approximately sodium carbonate and 0.2% sodium secondary alkyl sulfate surfactant in distilled water,

(0) twenty minutes after step (b) placing and mixing in the container 3.0 ml. of a mixture of phosphotungstic acid solution and approximately 0.1% hydrazine dihydrochloride,

(d) allowing the mixture of step (c) to sit for twenty minutes after the addition of the material of step (c) and then measuring the optical absorbance of the liquid in the container,

(e) placing in each of several transparent containers 0.1 m1. of different concentrations of uric acid standard containing a known concentration of uric acid,

(f) performing steps (b) through (d) with respect to each of the transparent containers of step (e),

(g) plotting a curve of optical absorbance versus concentration of uric acid in each of the containers of step (e), and

(h) comparing the optical absorbance of step (d) with the curve of step (g) to determine the concentration of uric acid in the blood serum, steps (a) through (d) being performed in any order with respect to steps (e) through (g).

11. A test system for the detection of uric acid in blood serum containing protein by comparing the optical absorbance of treated samples of uric acid standard with the optical absorbance of a treated sample of such blood serum, such test system comprising (a) a carbonate reagent including a solution of sodium carbonate and an alkyl sulfate surfactant and (b) a phosphotungstic reagent including phosphotungstic acid solution and hydrazine dihydrochloride.

12. The test system of claim -11 in which the sodium carbonate reagent includes approximately 5% sodium carbonate and 0.2% sodium secondary alkyl sulfate in distilled water.

13. The test system of claim 11 in which the hydrazine dihydrochloride is present in approximately 0.1%.

14. The test system of claim 12 in which the hydrazine dihydrochloride is present in approximately 0.1%.

References Cited UNITED STATES PATENTS 3,418,079 12/1968 Rey et al.

OTHER REFERENCES Martinek, R. G.: Journal of Clinical Pathology, vol. 18, pp. 777-9 (1965).

Simoes, M. S.: Arquivos Portugueses de Bioquimica, vol. 6, pp. 1927 (1962-3).

JOSEPH SCOVRO'NEK, Primary Examiner E. A. KATZ, Assistant Examiner US. Cl. X.R. 

